Listeria monocytogenes is a facultative intracytoplasmic pathogen which primarily infects pregnant women and immunocompromised individuals, including AIDS patients, and is a leading cause of death from food-borne illness. Murine listeriosis is a premier model for the study of cell- mediated immunity (CMI) in which antibody plays no role in protection. Previous studies have provided a molecular and cell biological explanation for the requirement for CMI, as intracytoplasmic bacteria are able to exploit a host system of actin-based motility to move from cell-to-cell without exposure to the humoral immune system. The primary bacterial determinant which governs cell-to-cell spread is ActA, a surface protein which mediates actin polymerization and profilin binding. ActA is a 70- kDa protein which contains four proline-rich repeats with the consensus sequence DFPPPPTDEEL. In Aim I, the role of the proline-rich repeats for ActA function will be characterized by successive deletion of individual repeats and by mutagenesis of residues within the repeats. Mutations will be constructed using PCR-based methods and introduced into the L. monocytogenes chromosome by allelic exchange. The mutants will be fully characterized for actin-based motility, profilin binding, actin nucleation, cell-to-cell spread, and virulence in mice. These studies will establish the role of the repeats and the importance of the prolines and charged residues within the repeat. ActA is phosphorylated inside of mammalian cells resulting in a dramatic shift in apparent molecular mass upon SDS-PAGE. In Aim II, the sites of phosphorylation will be identified by mutagenesis of serine and threonine residues, and the effect of these mutations will be fully evaluated in tissue culture models of infection. ActA will be phosphorylated in vitro using purified kinases in an attempt to reproduce its intracellular properties. In Aim III, the precise biochemical activity of Act A will be determined. First, a secreted form of ActA lacking its transmembrane domain will be purified to homogeneity using conventional methods of protein purification. Purified ActA and mutated derivatives, either soluble or coupled to sepharose, will be evaluated for actin nucleation and profilin binding. If ActA alone has no detectable activity, it will be activated by incubation in Xenopus egg and HeLa cell extracts. The component(s) of the extracts necessary for ActA activation will be identified and characterized. The proposed studies should further our understanding of an essential determinant of bacterial pathogenesis and basic mechanisms of host cell actin-based motility.